Long-acting vitamin b12



\ 2,920,015 nema ail, 5, 1960 LONG-ACTING VITAMIN 'Robert E. Thompson,:Momence, -Ill.,1assignor to Armour and Company, Chicago, 11]., acorporation of Illinois NmDrawing. Application August 27, *1957 SerialNo; 680,635

"4 Claims. [(Cl, 167-481) :and neurological .conditions and in =theprophylaxis of.

potential vitamin B deficiencies, such as .insgeriatric practice. In theparenteral administration of massive doses :of vitamin 18 :for thesepurposes the vitamin is iconventionally injected subcutaneously orintramuscularly in the form of an aqueous isotonic saline solution."Ihereupon the serum vitamin B is immediately elewated toatlevel-considerably in excess ofthe renal threshold for vitamin B andthe vitamin is rapidly excreted aine the urine within :a period of about24 :hours. .Consequentl-y, in order to provide increased availability of*vitamin B 'to :the body tissues in thesesituations .it is currentlynecessary to administer the vitamin to the :patient several times aweek. 1 I- have discovered a vitamin B composition which uponsubcutaneous or intramuscular injection provides a significant'elevationin the-serumavitamin B level which e'levationtcan be regulated such thatthe renal threshold for vitamin B -is not exceeded. Moreover, thiselevated serum-vitamin B level can be maintained -for a isignificantlyprolonged period of-time.

In accordance with this invention, these advantages are provided bya-v'itamin 'B c0mp0siti0n which includes la Nitamin B -active substancein combination with 'zinc :and Itannic acid. It :has been found thatthis composi- -tion produces upon subcutaneous or intramuscularinjection an elevation in the serum vitamin B level for a period as longas two weeks in comparison with the approximately 24 hour cluration of.efiectobtained with aqueous vitamin B preparations. 1

In the experimentation 'leading to the present invention, it wasobserved that the combination of vitamin "B -withzinc resulted in awater-soluble complex'which upon injection demonstrated resultscomparable -t o tho s'e obtained 'with aqueous vitamin B preparations,i.e., the zinc-vitamin B preparation-was rapidly excreted [in the urinewithin about 24 'hours a'fterinjection. Also it was found that thecombination of tannic acid-with vitamin B resulted in a preparationwhich upon injection increased the period during which the bloodlevelofvita- --min B -was elevated to slightly over 24 hours, but theprolongation of etfectwas in no way comparable'to'that obtained with'the-zinc-vitamin :B -tannate' composition of this-invention.Consequentlyythe combination of vitamin B with both zinc and tannic acidresults in a composition in which the zinc and tannic acid act'synergistically to efiect a prolongation of the --period during "whichthe serum vitamin B level is maintained at anelevatetl' level.Although-it=has not presently been possir "ble to elucidate the modeofaction by which'this response obtained,'-iti s"believed that thezinc-vitamin "B tannic acid composition, upon subcutaneous orintramuscnlar tannic acid, i.e., approximately moles of zinc ion per a mnistrat on o s, adep t of vi amii B12 at he S t 'of'injection from whichthe vitamin B .is metered into theblood stream at avrate such that theserum vitamin B is maintained at. a level above that which obtainedprior-to the injection thereof but below that of the renal thresholdandthat this metering effect'may be produced under appropriate conditionsfor a period as long as two weeks.

'The term vitamin B active substance conventionally 'reters to cobalaminor a compound structurally relatedto cobalamin which. promotes growth ofa micror m r ui n vitamin ,B12 o nu itio Su h a Lactobaci'llusleichmannii. This class of compounds includes such derivatives ofcobalamin as eyanocobalamin a d h o b lam o The omb n ion of thevitamig'Bm-act su ta with inc and tannic acid results in a complex which isinsoluble in water, and which maybe provided as an aqueous vitamin Bsuspension in accordance with this invention. That is, there is providedaccording to this invention a Water-insoluble complex having ascomponents thereof a vitaminB activeisubsjtance, zinc ion and tannicacid. The period of time during which the composit-ion produceselevation in the serum vitamin B le el, n t e t nt t w i hth sc um it m12 level is elevated during such'ip'eriod, may be controlled by la in te rat o o her th $1. m? c d r m b n d with t e itam lazt sfsi bs nsc ithis at rinsoluble' .complex. Furthermore, the amount of the vitamin'B'active substance included in this water-in- 'soluble complex will be adetermining factor in controllin th e od of me ur n w ic th rum vitaminB12.. v. 'i$ e t u on iw n t r s It has been found that about 1 mg. ofzinc ion is capable of completely insolubilizing in water 2 mgs. of puremole ,of tannic acid is required to provide an insoluble zinctannate.Also, it has been found that about 2 mgs. of pure tannic acid per mg. ofcrystallized cabalamin is capable of producing a completelywater-insoluble zincvitamin B -tannate preparation, i.e., 45 moles oftannie acid "are required per mole of vitamin B to obtain a completelywater-insoluble zinc vitarnin B -tannate composition. Thus, a completelywater-insoluble zinc-vitamin B -tannate may consist of approximately 1mg. of zinc ion, 2 mgs. of pure tannic acid and 1 mg; of crystallizedcobalamin. v p

'Ine'rnploying ratios of zinc and tannic acid less than would berequired to produce acompletely water-insoluble zinc-vitamin B -tannate,there will be obtained a preparation in which a portion of the vitamin Bis retained in the water-soluble form and in which such portion ofwater-soluble vitamin B behaves upon subcutaneous or intramuscularinjection in amanner similar to that of the conventional aqueousisotonic saline solution of vitamin B Thus, vitamin B compositions maybeobtained in accordance with this invention in which the ratio of zincand tannic acid to vitamin B can .be regulated to modify the intensityof eiiect upon injection, as well as the duration of time in which suchmodified intensity of effect is produced.

Although especially desirable results are achieved with the vitamin'Bcomposition of this invention when there is employed therein acrystalline vitamin B preparation, the advantages of the invention maybe obtained with any vitamin B preparation suitable for parenteraladministration. Also the zinc and tannic acid included in this vitamin Bcomposition should be suitia ble for parenteral administration.

In accordance with this "invention, the Vitamin B composition may beobtained by eombiningwith tannic acid 'and' vitamin B in an aqueousreaction medium a water-soluble zinc salt, such as zinc acetate and zincchloride, to obtain in the resulting aqueous suspension awater-insoluble zinc-vitamin B -tannate complex. In preparing aninjectible composition according to this process, there is formed asterile aqueous suspension of the water-insoluble zinc-vitamin B-tannate complex in an isotonic saline solution. This injectiblepreparation is obtained by combining a sterilized aqueous solution oftannic acid with a sterilized aqueous solution of crystalline vitamin Band thence aseptically combining with such sterile aqueous mixture ofvitamin B and tannic acid a sterilized aqueous solution of thewater-soluble zinc salt to obtain a sterile aqueous suspension of thewater-insoluble zinc-vitamin B -tannate complex.

In another aspect of this invention, it has been found that theinclusion of this water-insoluble Zinc-vitamin B -tannate complex in anaqueous gelatin solution results in an aqueous gelatin suspensionthereof which demonstrates upon subcutaneous or intramuscular injectionan elevation in the serum vitamin B level in excess of that obtainedwith an aqueous suspension of this complex without significantlyaltering the period of time during which thiselevation is obtained. Thegelatin employed in this aspect of the invention may be any gelatinsuitable for parenteral administration. Although the gelatin may beemployed in this aqueous gelatin suspension at any concentration lessthan 16 percent by weight, it has been found that an aqueous gelatinsuspension in which the gelatin concentration is from 0.5 to 6 percentby Weight produces the desired physiological efiect without renderingthe composition so viscous as to be injectible only with difi'iculty.Especially desirable results are obtained when the water-insolublezinc-vitamin B tannate complex is included in an aqueous gelatinsolution containing from 1 to 4 percent by weight gelatim Althoughthisaqueous suspension of the zinc-vitamin B -tannate complex issubstantially resistant to deterioration during prolonged storage, thetannic acid component of this complex is susceptible to oxidation withconsequent discoloration of the vitamin B composition. This tendency foroxidation of the tannic acid component of the complex may be avoided,and stability of the vitamin B composition during prolonged storage canbe substantially assured, in inhibiting oxidation thereof by partialdehydration of the aqueous medium in which the zinc-vitamin B -tannatecomplex is suspended. The partial dehydration of this aqueous suspensionmay be accomplished by including in the vitamin B composition a portionof for example propylene glycol or polyethylene glycol in the amount ofapproximately 50% by volume. Other substances which have been found toenhance the stability of this vitamin B composition includecarboxymethylcellulose and such reducing agents as cysteine and sodiumor potassium citrate.

Also, the zincvitamin B -tannate complex of this invention may beobtained in the form of a dry powder which can be reconstituted with anaqueous solution prior to injection. This embodiment may be produced bysubjecting to lyophilization under aseptic conditions a sterile aqueoussuspension of the zinc-vitamin B tannate complex. The resulting lyophileproduct can be reconstituted prior to injection with an aqueous isotonicsaline solution or an aqueous gelatin solution to obtain an aqueoussuspension of the zinc-vitamin B -tannate complex. The lyophile productdemonstrates exceptional stability during an extremely prolonged periodof storage, and the inclusion therein of a reducing agent, such ascysteine, and an antibacterial agent, such as parahydroxybenzoic acid,prevents deterioration and permits the reconstitution thereof in anaqueous solution without undesirable lumping or coagulating beingproduced in the resultant aqueous suspension.

In a further aspect of this invention, there is obtained in forming thewater-insoluble zinc-vitamin B -tannate complex with a relatively impurevitamin B preparation the selective solubilization of the impuritiesassociated with the vitamin B and consequent purification of the vitaminB composition. A method by which this purification of a relativelyimpure vitamin B preparation may be performed in the course of forming awaterinsoluble zinc-vitamin B -tannate complex is described hereinafterin Example X. The crude vitamin B preparations employed in thispurification process may be derived from animal sources, such as liver,and from microbiological sources according to methods well known in theart. 1

It will be apparent from the foregoing that especial advantages are tobe realized in the utilization of this zinc-vitamin B -tannate complexby subcutaneous or intramuscular injection. However, this vitamin Bcomposition may be orally administered to achieve beneficial results.

The advantages of this invention may be further illustrated by thefollowing specific examples:

Example I Tannic acid, in the amount of 550 mgs., was dissolved in 50ml. of distilled water, and the resulting solution was filtered througha sterilizing Selas candle into a sterile vessel. The Selas candle waswashed with an additional 12.5 ml. of water.

Crystalline cyanocobalamin, in the amount of 137.5 mgs., was dissolvedin 50 ml. of water, and the resulting solution was filtered through theSelas candle into the vessel containing the sterile tannic acidsolution. The Selas candle was then washed with an additional 12.5 ml.of water.

Crystalline zinc acetate, in the amount of 1.0 gms., was dissolved in 50ml. of water, and the resulting solution was filtered through the Selascandle into the vessel containing the sterile mixture of vitamin B andtannic acid to obtain an aqueous suspension of a zinc-vitamin B -tannatecomplex. The Selas candle was then washed with an additional 12.5 ml. ofwater.

Thereafter, an aqueous solution, in the amount of 50 ml., containing2.25 gms. of sodium chloride, mgs. of cysteine and 1.25 gms. of phenolwas filtered through the Selas candle into the vessel containing thesterile aqueous suspension of zinc-vitamin B -tannate complex, such thatthe volume of suspension in the sterile container was increased to atotal volume of 250 ml.

The isotonic suspension of the zinc-vitamin B -tannate complex therebyobtained, while undergoing continuous agitation, was filled asepticallyinto sterile 5 ml. pharmaceutical vials. The filled vials wereaseptically stoppered and sealed.

This vitamin B composition contained 500 mcg. of vitamin B per ml.

Example 11 The action of the vitamin B composition obtained according tothe method of Example I was compared with that of (1) an aqueousisotonic saline solution of vitamin B containing 0.5% of phenol and (2)an aqueous suspension of vitamin B -tannate prepared according to themethod of Example I except that the addition of the zinc acetate wasomitted.

A group of 8 rats was employed to test each of these vitamin Bcompositions. Each rat in the respective groups was injectedsubcutaneously with 1.0 ml. of the appropriate vitamin B composition,which was equivalent to 500 mcg. of vitamin B Theurine of each group ofrats was collected periodically and pooled. Merthio late was added tothe pooled urine to retard bacterial decomposition.

Water was provided tov the rats ad libitum throughout the period ofthese tests. When the urine of the rats was collected on successivedays, the rats were allowed food for a period of two hours each day. Onthe other hand, when the urine was collected. on alter= hate da'ysfoodwas providedadlibitum on such alternate days.

The. vitamin B content of the pooled urine was. determined by thechemical assay method of P. .1. Van

Melle published in the Journal of the American PharmaceuticalAssociation, January 1956.

The results obtained withjthe aqueous isotonic saline -solution ofvitamin B are presented in the following table, wherein the vitamin Bcontent of the urine is expressed as the total amount excreted by thegroup of test rats and as the calculated average value for each eat ofthe group.

Total amount Average of excreted amount of N 0,.of hours followinginjection vitamin B vitamin B pcr8 rats excreted per 1(m. meg.) rat (in.meg.)

The following results were obtained with the aqueous suspension of .B-tannate, in which the expression of re- It can be seen from theforegoing results that the absorption control obtained with the vitaminB -tannate composition is similar to that afforded by the aqueousvitamin B solution. I

The following results were obtained with the zincvitamin B -tannatecomposition obtained according to the method of Example I:

p i Total-amount- Average of excreted amount of No. of hoursfollowinginjectlon vitamin B vitamin B per 8 rats excreted per (111.meg.) rat (in. meg.)

It can be seen from the foregoing table that absorption control wasobtained with the zinc-vitamin B tannate composition for a period inexcess of 15'days,1whereas with the vitamin vB -tannate and aqueousvitamin B solution excretion of the vitamin B was substantially completeafter 72 hours. In each of the foregoing experiments the rats excretedno vitamin B during a 24 hour control ,period preceding the testsituation.

Example III The absorption control afforded by the vitamin B compositionobtained according to Example .I was com- .pared in humans with thatprovided "by. 'an aqueous isotonic saline solution of vitamin "B Each ofthese "vitamin B preparations wastested on a group of five patients.Each patient in the group was injected 'sub- :cutaneouSly with 1 ml. ofthe appropriate vitamin B rpreparationywhichxwas equivalent L .500 :mcg.:of vitamin 113. At selected intervals .after administration of thervitamin :B preparation :the content {Of -zv'itarnincB .in

the serum of each patient was determined aecording te themicrobiological assay method of Slteggs et a1.,"1 84,

The following results were obtained with the aqueous isotonic salinesolution of vitamin B wherein the vitamin B content of the serum isexpressed in 111. mcg. permL:

Time Interval After Injection (hours) Patient I 367 12,000 I. 793 1. 588461 437 233 17., 000 .1, 641 1, .426 315 216 338 13, 530 1,598 1,397 297245 484 L1. 880 1.665 :1, 554 .408 Z 1373 5.. .496 1,..850 1, 711 .1,502 577 r172 Average 384 I 17,560 1,683, 1,495; 412 359 The followingresults were obtained with the zincvitamin B -tannate composition,wherein thetabulated results are expressed in terms .of the serumvitamin B level in m. mcg. per ml.:

. Time.;l.-ntcrval.After Injection (hours) Patient H 7v 47 2 'sss- 1,519-2. 490 1, 502 1,319 .379 461; .1, 426 2,.5L3 v ,1, 426, .1, 367 '548513; 1 536 2, 669' 1, 490 *1, 717 466 ,1, 11-9 1 1, 542 1' 2, 513 l, 5481,349 980 .560. 1, 729 q .2, 700 1, 7.46, 1,100 Average... 569 648 1,1550' 2, 517 1,154.2 V 1,;522

It can be seen 'from =theiforegoi-ng that, .whereas in Ethe patientstreated with the :aqueous isotonic .salinesolution of vitamin B theserum vitamin B level returned to the normal value after about 44 hours,the patients treated with the zinc-vitamin .B -tannate compositiondemonstrated an elevated serum vitamin B level fora period in excess of216 hours.

Exnmple.-lV The urinary excretion of vitamin 613 "for "the patientstreated according to the method of Example 'III was determined, and theresults are expressed in the "following table in terms of mcg. ofvitamin B Time Interval After Injection (hours) Patient It can 'be seenfrom the foregoing, in substantiation of the conclusion expressed inExample :III, "that whereas absorption control was obtained withrthe.aqueousu'sotonic saline solution of vitamin B for a zperiodtof aboutp24hours, the absorption control afforded by thezincozitamin B -tannatecomposition .exceeded216 Ehouns.

Example '1 Another series of :human zpatients were treated With anaqueous isotonic saline :solution :of witaminxB .sand

the zinc-vitamin B -tannate composition obtained .ac-

cording to the method .of Example rI (in .the .manner 10f the experimentoutlined in Example III; J

The .zfdllowing :results were obtained :with -.the..'-aqueons isotonicsaline solution of vitamin B wherein the serum content of vitamin B isexpressed in 111. mcg. per ml.:

min B -tannate thereby obtained had the following composition:

I Tannic acid gms 1.1 Patient Time Interval After In ection (hours) 5Vitamin B12 mg 275 Zinc acetate gms 2.0 0 s 24 72 144 216 288 To thissterile suspension of zinc-vitamin B -tannate 344 mm um 945 676 519there was added a sterile-filtered solution containing 600 2g; 3:388figg gig g3 mg. of cysteine and 500mg. of methyl parahydroxy- 297 7,3002,900 1,280 700 577 335 benzolc acid. The resulting suspension wasdiluted to 233 6,700 940 566 530 414 373 a final volume of 250 ml. withsterile-filtered water. Average 296 6,800 1,773 835 699 543 433 Thisdilute sterile suspension was aseptically filled into pharmaceuticalvials adapted for the purpose of lyophil- The following results wereobtained with the zinc-vitaization, the amount 25 of suspension P min B-tannate composition obtained according to the 5 6 The fined vials wereaseptlcifllly Stoppered method of Example I, wherein the serum contentof vitaand Q contents thereof frozen- T of frozen min B is expressed inm. mcg. per ml.: material were SllbJCCtfid to a lyophilizationoperation.

This lyophile zinc-vitamin B -tannate composition Time Interval AfterInjection (hours) thereby obtained can be reconstituted with water or anPatient 20 aqueous isotonic saline solution prior to nnectlon.

0 8 24 72 144 216 288 This lyophile preparation demonstrates anindefinite shelf life, and therefore is especially suitable for those869 1,970 L560 1,569 2,070 2 740 2157 purposes in which the zinc-vitaminB -tannate composi- 402 1,050 650 1,100 1, 019 1101s '75s tion is storedfor a prolonged period of time prior to is 1,123 1; is: i193; in 1,83825 245 1,143 700 556 799 910 805 Example VIII Average 471 1.271 8541.149 1,563 1,461 .0 The lyophile preparation of Example VII wasreconstituted with an aqueous isotonic saline solution contain- Theforegoing tabulated results show that while the ing 1% of partiallyhydrolyzed pharmaceutical gelatin. serum content of vitamin B in thepatients treated with This aqueous gelatin suspension of thezinc-vitamin the aqueous solution of vitamin B remained at an ele- B-tannate, upon subcutaneous or intramuscular injecvated level for aperiod of about 144 hours, the serum tion, produces an elevation of theserum vitamin B content of vitamin B of those patients treated with thelevel in excess of that obtained with an aqueous suspenzinc-vitamin B-tannate composition was maintained at or sion of the zinc-vitamin B-tannate composition. Also, a significantly elevated level for a periodin excess of there is obtained a duration of effect in excess of 216 216hours. hours.

Example VI Example IX The urine of patients treated according to thepro- The degree of elevation of the serum vitamin B level, cedure ofExample V was also subjected to vitamin B and the period of time duringwhich the serum vitamin determinations in the manner of Example IV. Blevel is maintained at the elevated value, may be The results obtainedwith the patients treated with the controlled by regulating the ratio oftannic acid and aqueous isotonic saline solution of vitamin B are shownzinc to the vitamin B in the zinc-vitamin B -tannate in the followingtable, wherein the urine content of vitacomposition. min B is expressedin mcg.: If the ratio of vitamin B to tannic acid and zinc is TimeInterval After Injection (hours) Patient Avera 0. 320 0. 353 0. 104

It can be seen from the foregoing results that the urinary excretion ofvitamin B was substantially complete Within 24 hours in the patientstreated with the aqueous vitamin B solution, while that of the patientstreated with the zinc-vitamin B -tannate composition was at a minimallevel even after a period of 168 hours.

Example VI] A sterile dry preparation of the zinc-vitamin B -tannatecomposition was obtained by the following method:

Aqueous solutions of tannic acid, crystalline cyanocobalamin and zincacetate were sterile-filtered and combined in a sterile containeraccording to the method of -Example I. The sterile aqueous suspension ofzinc-vitadoubled over the relative values employed in the method ofExample I, there is obtained an aqueous suspension of the zinc-vitamin B-tannate complex having the fol- This modified preparation, uponsubcutaneous or intramuscular injection, will demonstrate an elevationin the serum vitamin B level exceeding that shown in Examples II to VIIfor the preparation obtained by the method of Example I, while-theperiod of time during 9 which this elevation in the serum vitamin Blevel is produced will be correspondingly reduced Example X A relativelycrude preparation of vitamin B can be employed in a method substantiallyaccording to that set forth in Example I to achieve a zinc-vitamin Btannate composition suitable for parenteral administration, whereinpurification of the vitamin B is achieved in the course of forming theinsoluble zinc-vitamin B tannate complex.

In this modified procedure, an aqueous solution of relatively impurevitamin B is produced, containing 100 mcg. of vitamin B activity per ml.To this solution is added zinc acetate in such amount as to provide inthe resulting solution a zinc ion concentration of 200 mcg. per ml. ThepH of this solution is adjusted to 7.5 with sodium hydroxide, and thensuch solution is filtered to separate the zinc-precipitated impuritiesfrom the soluble vitamin B activity.

To the filtered solution is added tannic acid in an amount such that amaximum of vitamin B impurities are precipitated while retainingsubstantially all of the vitamin B activity in the soluble form. Theresulting tannic acid precipitate is separated from the vitamin Bsolution by filtration and discarded.

Thereafter, tannic acid is added to the vitamin B containing filtrate inan amount equivalent to 2 mg. of tannic acid per mg. of vitamin Btherein. Then there is added to the filtrate zinc acetate in such amountas to obtain in the resulting suspension 1 mg. of zinc ion per 2 mg. oftannic acid. The resulting zinc-vitamin B -tannate precipitate isseparated from the supernatant liquid by filtration or centrifugation.

The separatedzinc-vitamin B -tannate precipitate may be suspended inwater and reconstituted as an aqueous solution by acidification withcitric or hydrochloric acid. The reconstituted solution may besterile-filtered, and such sterile solution may be reprecipitated by theaddition thereto of a sterile solution of sodium hydroxide or sodiumphosphate.

In the alternative, the separated zinc-vitamin B -tam nate compositionmay be mixed with water to form an aqueous suspension thereof andsterilized by autoclaving in the presence of cysteine to obtain apreparation suitable for parenteral administration.

While in the foregoing specification this invention has been describedin considerable detail by references to specific embodiments thereof, itwill be apparent to those skilled in the art that the invention-issusceptible to other embodiments and that many of the details set forththerein can be varied widely .without departing from the spirit andconcept of the invention.

I claim:

1. An injectible vitamin B composition, comprising a water-insolublecomplex having a vitamin B -active substance component, a 'zinccomponent and a tannic acid component.

2. An injectible vitamin B composition, comprising an aqueous suspensionof a water-insoluble complex having a vitamin-B -active substancecomponent, a zinc component and a tannic acid component.

3. An injectible vitamin B composition, comprising an aqueous gelatinsuspension of a'water-insoluble complex having a vitamin B -activesubstance component, a zinc component and a tannic acid component.

4. In a method of selectively separating vitamin B from associatedimpurities, the steps of-combining in an aqueous solution with a crudevitamin B preparation containing a vitamin B -active substance andassociated impurities a water-soluble zinc salt and tannic acid toobtain in the resulting aqueous suspension a water-insoluble complexhaving a vitamin B -active substance component, a zinc component and atannic acid component and to selectively solubilize said impurities, andseparating said water-insoluble complex from the supernatant liquid.

References Cited in the file of this patent UNITED STATES PATENTS2,621,144 Holland Dec. 9, 1952

1. AN INJECTIBLE VITAMIN B12 COMPOSITION, COMPRISING A WATER-INSOLUBLECOMPLEX HAVING A VITAMIN B12-ACTIVE SUBSTANCE COMPONENT, A ZINCCOMPONENT AND A TANNIC ACID COMPONENT.
 4. IN A METHOD OF SELECTIVELYSEPARATING VITAMIN B12 FROM ASSOCIATED IMPURITIES, THE STEPS OFCOMBINING IN AN AQUEOUS SOLUTION WITH A CRUDE VITAMIN B12 PREPARATIONCONTAINING A VITAMIN B12-ACTIVE SUBSTANCE AND ASSOCIATED IMPURITIES AWATER-SOLUBLE ZINC SALT AND TANNIC ACID TO OBTAIN IN THE RESULTINGAQUEOUS SUSPENSION A WATER-IN SOLUBLE COMPLEX HAVING A VITAMINB12-ACTIVE SUBSTANCE COMPONENT, A ZINC COMPONENT AND A TANNIC ACIDCOMPONENT AND TO SELECTIVELY SOLUBLIZE SAID IMPURITIES, AND SEPARATINGSAID WATER-INSOLUBLE COMPLEX FROM THE SUPERNATANT LIQUID.